In the past decade, applications of spectroscopy and microspectroscopy have greatly advanced into areas of clinical study. The potential of various spectroscopic techniques, including mid-infrared spectroscopy, for screening and disease diagnosis in clinical settings has been investigated. Research has been conducted on samples from a variety of organs and cellular types. The results of those studies have generally confirmed that infrared spectroscopy can differentiate malignant from normal cells.
As is well known, infrared spectroscopy is typically performed in either of transmission or reflection modes. The energy range of the infrared light used is typically in the mid-infrared range of about 4000 cm.sup.-1 to 400 cm.sup.-1. When operating in the transmission mode, the infrared beam passes through the sample once and is detected. When the energy incident to the sample matches an excitation energy of the sample material, energy is absorbed and the intensity of the incident radiation is reduced as a result. A detector measures the intensity of the transmitted light as a function of energy for identifying the energy absorbed. When operating in the reflection mode, the sample is placed on a reflective substrate such as a thin metal layer that does not absorb the incident infrared energy. The incident beam passes through the sample as for a transmission measurement but is then reflected off the substrate and passes through the sample a second time before it is detected. As for the transmission mode, energy from the incident beam is absorbed when the energy of the beam matches an excitation energy of the sample material and, as a result, the intensity of the incident light is reduced.
Samples for mid-infrared spectroscopy are conventionally prepared in a manner that is incompatible with that commonly used in a pathology laboratory. Typically, mid-infrared measurements are carried out on thin sections of microtomed tissue placed between two windows of infrared-transparent material, such as BaF.sub.2 or CaF.sub.2, or in a diamond anvil cell. Other studies involve the suspension of cells, centrifugation of cells into a pellet, extraction of DNA from tissues, or measurement of biological fluids in solution.
More recently, infrared microspectroscopy has been used in the study of such samples. This technique enables measurement of microscopic tissue areas and mapping of the whole tissue with the capability of visualizing the areas measured. Samples for infrared microspectroscopy are conventionally placed on an infrared-transparent material, such as BaF.sub.2 or CaF.sub.2 windows, for transmission measurements, or on gold-coated slides for reflection measurements. Gold provides a good reflective surface so that gold slides are the conventional choice for infrared reflection studies.
The results of the spectroscopic measurements are typically compared to the results of a study by a pathologist on a separate sample from the same source for classification. A pathologist uses visible microscopy to analyze a biological sample. The sample for pathologic study is conventionally a thin section fixed on a glass slide and stained. Glass slides, which are transparent to visible light, are inexpensive and easy to work with. In contrast, a gold coated slide typically costs several times more than a plain glass slide. Plain glass, however, is strongly absorptive in the mid-infrared range. Thus, when coupled with conventional infrared spectroscopic techniques, the glass absorption would be expected to overwhelm the spectral response from the sample in transmission measurements. A plain glass slide is also unsuitable for reflection measurements because it does not provide a good reflective surface. Significantly, samples prepared for standard pathologic studies are generally deemed unsuitable for infrared measurements due to the use of a plain glass slide as the supporting substrate.
Moreover, regardless of its exact method of preparation, a sample prepared in the conventional manner for infrared studies is damaged such that it cannot be recovered for examination by a pathologist to verify the diagnoses based on the infrared measurements or to perform any other pathologic study. At the same time, older samples mounted on glass slides and preserved for archival purposes cannot be subjects for analysis by conventional infrared spectroscopic techniques. The use of different samples prepared in different ways for spectroscopic and conventional pathologic studies also inevitably introduces some unreliability in the comparison of the results from the two different studies.